Anti-Inflammatory Pyrazolopyrimndines

ABSTRACT

The present invention provides anti-inflammatory compounds useful in the treatment of diseases and conditions in which inflammation is involved in disease progression or the manifestation of symptoms of the disease or condition.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/719,722, filed Dec. 5, 2008, which claims benefit of PCT/US2005/42524filed Nov. 21, 2005, which claims the benefit of U.S. Provisional PatentApplication No. 60/629,639, filed Nov. 19, 2004, each of which isincorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant AI 044009awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Protein kinases are involved in a wide variety of cellular processes,such as growth factor response, cytokine response, immune response,stress response, and cell cycle regulation, to specify only a few suchprocesses. Because their improper regulation is believed to causevarious diseases including cancer and inflammation, protein kinases areimportant drug targets for the treatment of these diseases.

Inflammatory conditions, particularly chronic inflammatory diseases, areof particular interest to developers of new pharmaceutical products,because such conditions and diseases are widespread, and improvedtherapies for them are needed. These diseases are caused by action ofthe immune system, including the inappropriate activation of T cells,expression of regulatory cytokines and chemokines, loss of immunetolerance, and the like. Modulation of the immune response varies withthe specific factors produced and the receptors present on theresponding cell. Among these diseases are autoimmune and/or chronicinflammatory diseases, which include multiple sclerosis and inflammatorybowel diseases (“IBD,” including ulcerative colitis and Crohn'sdisease), colitis, diseases of the joints, such as rheumatoid arthritis,diseases involving the destruction or improper alteration of nucleicacids, as observed with systemic lupus erythematosus and other diseasessuch as psoriasis, insulin dependent diabetes mellitus (IDDM), Sjogren'sdisease, myasthenia gravis, thyroid disease, Alzheimer's disease,uveitis, and cardiovascular disease.

Thus, there is a need in the art for effective anti-inflammatorycompounds useful in the treatment of disease. The present inventionaddresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides anti-inflammatory compounds useful in thetreatment of diseases and conditions in which inflammation is involvedin disease progression or the manifestation of symptoms of the diseaseor condition.

In one aspect, the present invention provides a compound represented bythe following structural formula:

In Formula (I) above, R¹ is selected from substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

R², R³, and R⁴ are, independently, hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R³ and R⁴ are optionally joined with thenitrogen to which they are attached to form a substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl.

L¹ is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted cycloalkylene, or substituted or unsubstitutedheteroalkylene.

In another aspect, the present invention provides a method for treatingor preventing a disorder characterized by abnormal inflammation, saidmethod including administering to a subject a therapeutically effectiveamount of a compound of the present invention.

In another aspect, the present invention provides pharmaceuticalformulations of a compound of the present invention, and methods forusing such compounds and their pharmaceutical formulations in thetreatment of inflammatory diseases and conditions.

In another aspect, the present invention provides methods for making thecompounds and pharmaceutical formulations of the invention.

In another aspect, the present invention provides compounds andpharmaceutical formulations for use in the treatment of an inflammatorydisease or condition.

In certain embodiments, the compounds of the invention are T cellactivation inhibitors. In another embodiment, the compounds of theinvention are monocyte activation inhibitors. In another embodiment, thecompounds of the invention are gamma interferon signaling inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates four exemplary BioMAP™ (BioSeek, Inc., Burlingame,Calif.) inflammation model systems useful in detecting anddiscriminating modifiers of multiple targets and pathways, where panel(A) sets forth multiple therapeutically relevant targets and pathwaysthat are detected and discriminated in BioMAP models, including a largenumber of kinases, and panel (B) provides details on the four exemplarymodel systems.

FIG. 2 illustrates the decrease in the number of recruited leukocytes ina mouse model of peritoneal inflammation resulting from theadministration of an anti-inflammatory pyrazolopyrimidine of the presentinvention.

FIG. 3 illustrates the decrease in macrophage and neutrophil recruitmentin a mouse model of peritoneal inflammation resulting from theadministration of an anti-inflammatory pyrazolopyrimidine of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Abbreviations used herein have their conventional meaning within thechemical and biological arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e. unbranched) or branched chain,or cyclic hydrocarbon radical, or combination thereof, which may befully saturated, or mono- or polyunsaturated, and can include di- andmultivalent radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include, but are not limited to, groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, and homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds or both. Examples of unsaturated alkyl groups include, but are notlimited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, and3-butynyl, as well as the higher homologs and isomers. Alkyl groups thatare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene,” by itself or as part of another substituent, meansa divalent radical derived from an alkyl, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combinations thereof, consisting of at least onecarbon atom and at least one heteroatom selected from the groupconsisting of O, N, P, Si and S, and wherein the nitrogen and sulfuratoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N, P, S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃, O—CH₃, —O—CH₂—CH₃, and —CN. Up to two heteroatoms maybe consecutive, such as, for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃.Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means a divalent radical derived from heteroalkyl, asexemplified, but not limited to, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—. As described above, heteroalkylgroups, as used herein, include those groups that are attached to theremainder of the molecule through a heteroatom, such as —C(O)R′,—C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” isrecited, followed by recitations of specific heteroalkyl groups, such as—NR′R″ or the like, it will be understood that the terms heteroalkyl and—NR′R″ are not redundant or mutually exclusive. Rather, the specificheteroalkyl groups are recited to add clarity. Thus, the term“heteroalkyl” should not be interpreted as used herein as excludingspecific heteroalkyl groups, such as —NR′R″ or the like.

The term “alkoxy” refers to an alkyl attached to the remainder of themolecule via an oxygen heteroatom. The alkyl portion of the alkoxy groupmay be any appropriate length. In some embodiments, the alkyl portion ofthe alkoxy group is a C₁-C₂₀ alkyl (i.e. C₁-C₂₀ alkoxy). In someembodiments, the alkyl portion of the alkoxy group is a C₁-C₁₀ alkyl(i.e. C₁-C₁₀ alkoxy). In some embodiments, the alkyl portion of thealkoxy group is a C₁-C₁₀ alkyl (i.e. C₁-C₁₀ alkoxy).

An “alkylesteryl,” as used herein, refers to a moiety having the formulaR′—C(O)O—R″, wherein R′ is an alkylene moiety and R″ is an alkyl moiety.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The terms “cycloalkylene”and “heterocycloalkylene” refer to the divalent derivatives ofcycloalkyl and heterocycloalkyl, respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (preferably from 1 to 3 rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to four heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a carbon or heteroatom.Non-limiting examples of aryl and heteroaryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryland heteroaryl ring systems are selected from the group of acceptablesubstituents described below. The terms “arylene” and “heteroarylene”refer to the divalent derivatives of aryl and heteroaryl, respectively.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above in combination with another moiety. Thus, theterm “arylalkyl” is meant to include those radicals in which an arylgroup is attached to an alkyl group (e.g., benzyl, phenethyl,pyridylmethyl and the like) including those alkyl groups in which acarbon atom (e.g., a methylene group) has been replaced by, for example,an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl,3-(1-naphthyloxy)propyl, and the like). However, the term “haloaryl,” asused herein is meant to cover only aryls substituted with one or morehalogens.

The term “oxo” as used herein means an oxygen that is double bonded to acarbon atom.

Each of above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and“heterocycloalkyl”, “aryl,” “heteroaryl” as well as their divalentradical derivatives) are meant to include both substituted andunsubstituted forms of the indicated radical. Preferred substituents foreach type of radical are provided below.

Substituents for alkyl, heteroalkyl, cycloalkyl, heterocycloalkylmonovalent and divalent derivative radicals (including those groupsoften referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl,alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, —OC(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g.,aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR/R″ is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin art will understand that the term “alkyl” is meant to include groupsin which carbon atoms are bound to groups other than hydrogen groups,such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃,—C(O)CF₃, —C(O)CH₂OCH₃), and the like.

Similar to the substituents described for alkyl radicals above,exemplary substituents for aryl and heteroaryl groups (as well as theirdivalent derivatives) are varied and are selected from, for example andwithout limitation: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′,—C(O)R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″,—NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, —NRSO₂R′, —CN and —NO₂, —R′, —N₃, —CH(Ph)₂,fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl, in a number, when present,ranging from one to the total number of open valences on aromatic ringsystem; and where R′, R″, R′″ and R″″ are independently selected fromhydrogen, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present.

Two of the substituents on adjacent atoms of an aryl or heteroaryl ringmay optionally form a ring of the formula —T—C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′— or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)₂—, —S(O)₂NR′— or a singlebond, and r is an integer of from 1 to 4. One of the single bonds of thenew ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of an aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′—(C″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″ and R′″ are independentlyselected from hydrogen, substituted or unsubstituted alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.

As used herein, the term “heteroatom” or “ring heteroatom” is meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A)-OH, —NH₂, —SH, —CN, —CF₃, —NO₂, oxo, halogen, unsubstituted        alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,        unsubstituted heterocycloalkyl, unsubstituted aryl,        unsubstituted heteroaryl, and    -   (B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, and        heteroaryl, substituted with at least one substituent selected        from:        -   (i) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,            unsubstituted alkyl, unsubstituted heteroalkyl,            unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,            unsubstituted aryl, unsubstituted heteroaryl, and        -   (ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,            and heteroaryl, substituted with at least one substituent            selected from:            -   (a) oxo, —OH, —NH₂, —SH, —CN, —CF₃, —NO₂, halogen,                unsubstituted alkyl, unsubstituted heteroalkyl,                unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, unsubstituted                heteroaryl, and            -   (b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,                aryl, or heteroaryl, substituted with at least one                substituent selected from oxo, —OH, —NH₂, —SH, —CN,                —CF₃, —NO₂, halogen, unsubstituted alkyl, unsubstituted                heteroalkyl, unsubstituted cycloalkyl, unsubstituted                heterocycloalkyl, unsubstituted aryl, and unsubstituted                heteroaryl.

A “size-limited substituent” or “size-limited substituent group,” asused herein means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 4 to 8 membered heterocycloalkyl.

A “lower substituent” or “lower substituent group,” as used herein meansa group selected from the substituents described above for a“substituent group,” wherein each substituted or unsubstituted alkyl isa substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, and each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl.

The compounds of the present invention may exist as salts. Accordingly,the present invention includes such salts. Examples of applicable saltforms include hydrochlorides, hydrobromides, sulfates,methanesulfonates, nitrates, maleates, acetates, citrates, fumarates,tartrates (e.g., (+)-tartrates, (−)-tartrates or mixtures thereofincluding racemic mixtures, succinates, benzoates and salts with aminoacids such as glutamic acid. These salts may be prepared by methodsknown to those skilled in art. Also included are base addition saltssuch as sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of acceptable acid addition salts include thosederived from inorganic acids like hydrochloric, hydrobromic, nitric,carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric,dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, orphosphorous acids and the like, as well as the salts derived organicacids like acetic, propionic, isobutyric, maleic, malonic, benzoic,succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike. Certain specific compounds of the present invention contain bothbasic and acidic functionalities that allow the compounds to beconverted into either base or acid addition salts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are in any eventencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent or otherwisesuitable for the uses contemplated by the present invention and arewithin the scope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the enantiomers, racemates,diastereomers, tautomers, geometric isomers, stereoisometric forms thatmay be defined, in terms of absolute stereochemistry, as (R)- or (S)-or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those which are known in art to betoo unstable to synthesize and/or isolate. The present inventionincludes compounds in their racemic and optically pure forms. Opticallyactive (R)- and (S)-, or (D)- and (L)-isomers may be prepared usingchiral synthons or chiral reagents, or resolved from mixtures usingconventional techniques. When the compounds described herein containolefinic bonds or other centers of geometric asymmetry, and unlessspecified otherwise, it is intended that the compounds include both Eand Z geometric isomers.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of atoms that constitutesuch compounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are encompassedwithin the scope of the present invention.

The term “pharmaceutically acceptable salts” is meant to include saltsof active compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituent moieties found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired pharmaceutically acceptable base,either neat or in a suitable inert solvent. Examples of pharmaceuticallyacceptable base addition salts include sodium, potassium, calcium,ammonium, organic amino, or magnesium salt, or a similar salt. Whencompounds of the present invention contain relatively basicfunctionalities, acid addition salts can be obtained by contacting theneutral form of such compounds with a sufficient amount of the desiredpharmaceutically acceptable acid, either neat or in a suitable inertsolvent. Examples of pharmaceutically acceptable acid addition saltsinclude those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention or one or more of their active metabolites. Additionally,prodrugs can be converted to the compounds of the present invention bychemical or biochemical methods in an ex vivo environment. For example,prodrugs can be slowly converted to the compounds of the presentinvention when placed in a transdermal patch reservoir with a suitableenzyme or chemical reagent.

I. Anti-Inflammatory Compounds

In one aspect, the present invention provides a compound represented bythe following structural formula:

In Formula (I) above, R¹ is selected from substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

R², R³, and R⁴ are, independently, hydrogen, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. R³ and R⁴ are optionally joined with thenitrogen to which they are attached to form a substituted orunsubstituted heterocycloalkyl or substituted or unsubstitutedheteroaryl.

L¹ is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted cycloalkylene, or substituted or unsubstitutedheteroalkylene. In some embodiments, where L¹ is a bond, R² is t-butyl,and R³ and R⁴ are hydrogen, then R¹ is not para-methylphenyl. In someembodiments, where L¹ is a bond, R² is unsubstituted alkyl, and R² andR³ are hydrogen, then R¹ is not methylphenyl.

In some embodiments, the compounds of Formula I do not include thosecompounds set forth in U.S. Pat. No. 5,593,997, U.S. Pat. No. 6,383,790,U.S. Pat. No. 5,981,533, U.S. Pat. No. 6,521,417, U.S. Pat. No.6,921,763, U.S. Pat. No. 6,713,474, U.S. Pat. No. 6,660,744, USPApplication 2002/0156081, USP Application 2003/0073218, USP Application2005/0085472, each of which are incorporated by reference in theirentirety for all purposes. In some embodiments, the compounds of theinvention are compounds other than the specific compounds set forth inthe aforementioned patents and published patent applications.

In some embodiments, the compounds of the invention are the subset ofcompounds of the compounds of Formula (I) that do not displacestaurosporine from a protein kinase where the compound is present at aconcentration of less than or equal to 10 μM. In some embodiments, thecompounds of the invention are the subset of compounds of the compoundsof Formula (I) that do not inhibit or significantly decrease proteinkinase activity when contacted with the kinase at a concentration ofless than or equal to 10 μM. In some related embodiments, the compoundsof the invention are the subset of compounds of the compounds of Formula(I) that do not decrease protein kinase activity more than 1%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, relative to the kinaseactivity in the absence of the compound. In some related embodiments,the compound that does not displace staurosporine as described aboveand/or does not inhibit or substantially decrease the activity of aprotein kinase as described above, is a compound of Formula (I) whereinR¹ is a substituted phenyl (e.g. substituted with a halogen), R³ and R⁴are hydrogen or unsubstituted C₁-C₁₀ alkyl, L¹ is a bond orunsubstituted C₁-C₁₀ alkylene, and R² is unsubstituted C₁-C₁₀ alkyl. Ina further related embodiment, R¹ is a halophenyl, R³ and R⁴ are hydrogenor methyl, L¹ is unsubstituted C₁-C₁₀ alkylene, and R² is unsubstitutedisopropyl. In a still further related embodiment, R³ is methyl, R₃ ishydrogen, and L¹ is methylene. In some embodiments, the compound iscompound 76 of Table 1, below.

In some embodiments, R¹ is substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

R¹ may also be an unsubstituted C₃-C₈ cycloalkyl, unsubstituted 4 to 8membered heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.Additionally, R¹ may be C₃-C_(s) cycloalkyl, 4 to 8 memberedheterocycloalkyl, aryl, or heteroaryl, that is substituted with asubstituent selected from a halogen (e.g. fluorine and/or chlorine),hydroxyl, sulfhydryl, cyano, nitro, unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₈cycloalkyl, unsubstituted 4 to 8 membered heterocycloalkyl,unsubstituted aryl, and unsubstituted heteroaryl. In some embodiments,R¹ is substituted or unsubstituted aryl (e.g. phenyl), or substituted orunsubstituted heteroaryl (e.g. benzothiophenyl).

In some embodiments, R¹ is a substituted phenyl having the formula:

In addition to a substituted phenyl of Formula (II), R¹ may be asubstituted or unsubstituted benzodioxolanyl, substituted orunsubstituted acenaphthenyl, substituted or unsubstituted naphthyl,substituted or unsubstituted benzothiophenyl, substituted orunsubstituted cyclopentyl, or substituted or unsubstituted thienyl. In arelated embodiment, the substituted or unsubstituted naphthyl is asubstituted or unsubstituted naphthalen-2-yl. Alternatively, thesubstituted or unsubstituted naphthyl may be a naphthalen-1-ylsubstituted with a substituent selected from a hydroxyl, sulfhydryl,cyano, nitro, substituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl, substituted thienyl, orunsubstituted cyclopentyl.

In some embodiments, the naphthalen-1-yl substituent is a hydroxyl,sulfhydryl, cyano, nitro, substituted C₁-C₂₀ alkyl, substituted orunsubstituted 2 to 20 membered heteroalkyl, substituted or unsubstitutedC₃-C₈ cycloalkyl, substituted or unsubstituted 4 to 8 memberedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; thienyl substituted with a substituted orunsubstituted alkyl; or unsubstituted cyclopentyl.

In other embodiments, R¹ is unsubstituted benzodioxolanyl orbenzodioxolanyl substituted with a substituent selected from a halogen,hydroxyl, sulfhydryl, cyano, nitro, unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₈cycloalkyl, unsubstituted 4 to 8 membered heterocycloalkyl,unsubstituted aryl, and unsubstituted heteroaryl.

Alternatively, R¹ is unsubstituted acenaphthenyl or acenaphthenylsubstituted with a substituent selected from a halogen, hydroxyl,sulfhydryl, cyano, nitro, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to10 membered heteroalkyl, unsubstituted C₃-C₈ cycloalkyl, unsubstituted 4to 8 membered heterocycloalkyl, unsubstituted aryl, and unsubstitutedheteroaryl.

R¹ may also be a unsubstituted naphthalen-2-yl or naphthalen-2-ylsubstituted with a substituent selected from a halogen, hydroxyl,sulfhydryl, cyano, nitro, unsubstituted alkyl, unsubstituted 2 to 10membered heteroalkyl, unsubstituted C₃-C₈ cycloalkyl, unsubstituted 4 to8 membered heterocycloalkyl, unsubstituted aryl, and unsubstitutedheteroaryl.

In some embodiments, R¹ is naphthalen-1-yl substituted with asubstituent selected from a hydroxyl, sulfhydryl, cyano, nitro,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₈cycloalkyl, unsubstituted 4 to 8 membered heterocycloalkyl,unsubstituted aryl, and unsubstituted heteroaryl. The naphthalen-1-ylsubstituent may also be selected from C₁-C₁₀ alkyl, 2 to 10 memberedheteroalkyl, C₃-C₈ cycloalkyl, 4 to 8 membered heterocycloalkyl, aryl,or heteroaryl, that is substituted with a substituent selected from ahalogen, hydroxyl, sulfhydryl, cyano, nitro, unsubstituted C₁-C₁₀ alkyl,unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₈cycloalkyl, unsubstituted 4 to 8 membered heterocycloalkyl,unsubstituted aryl, and unsubstituted heteroaryl.

In other embodiments, R¹ is thienyl substituted with an unsubstitutedC₁-C₁₀ alkyl or a C₁-C₁₀ alkyl substituted with a substituent selectedfrom a halogen, hydroxyl, sulfhydryl, cyano, nitro, unsubstituted C₁-C₁₀alkyl, unsubstituted 2 to 10 membered heteroalkyl, unsubstituted C₃-C₈cycloalkyl, unsubstituted 4 to 8 membered heterocycloalkyl,unsubstituted aryl, and unsubstituted heteroaryl.

R¹ may also be an unsubstituted cyclopentyl. Alternatively, R¹ isunsubstituted benzodioxolanyl or benzodioxolanyl substituted with asubstituent selected from a halogen and unsubstituted C₁-C₁₀ alkyl;unsubstituted acenaphthenyl or acenaphthenyl substituted with asubstituent selected from a halogen and unsubstituted C₁-C₁₀ alkyl;unsubstituted naphthalen-2-yl or naphthalen-2-yl substituted with asubstituent selected from a halogen and unsubstituted C₁-C₁₀ alkyl; orthienyl substituted with an unsubstituted C₁-C₁₀ alkyl or a C₁-C₁₀ alkylsubstituted with a substituent selected from a halogen and unsubstitutedC₁-C₂₀ alkyl; or unsubstituted cyclopentyl. R¹ may additionally be anaphthalen-1-yl substituted with an unsubstituted 2 to 10 memberedheteroalkyl; or C₁-C₁₀ alkyl, or 2 to 10 membered heteroalkylsubstituted with a substituent selected from a halogen and unsubstitutedC₁-C₂₀ alkyl.

In some embodiments, R¹ is unsubstituted benzodioxolanyl; unsubstitutedacenaphthenyl; unsubstituted naphthalen-2-yl; naphthalen-2-ylsubstituted with a substituent selected from a halogen and unsubstitutedC₁-C₅ alkyl; or thienyl substituted with an unsubstituted C₁-C₅ alkyl;or unsubstituted cyclopentyl. R¹ may additionally be naphthalen-1-ylsubstituted with an unsubstituted 2 to 5 membered heteroalkyl; or C₁-C₅alkyl or 2 to 5 membered heteroalkyl, that is substituted with a halogenand unsubstituted C₁-C₂₀ alkyl.

In other embodiments, R¹ is unsubstituted benzodioxolanyl; unsubstitutedacenaphthenyl; unsubstituted naphthalen-2-yl or naphthalen-2-ylsubstituted with a halogen; thienyl substituted with an unsubstitutedC₁-C₅ alkyl; or unsubstituted cyclopentyl.

In some embodiments, R¹ is R¹⁴-substituted C₁-C₂₀ alkyl, 2 to 20membered unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl,R¹⁵-substituted heteroaryl, substituted or unsubstituted fused-ringheteroaryl, or R¹⁶-substituted aryl. R¹⁴ is oxo, —OH, halogen, —CF₃,—NH₂, 2 to 20 membered substituted or unsubstituted heteroalkyl (e.g.alkoxy), 3 to 7 membered substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted heteroaryl, or R¹⁶-substituted aryl. R¹⁵ is—OH, halogen, —CF₃, —NH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl. R¹⁶is substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heteroaryl, orsubstituted or unsubstituted aryl, or substituted or unsubstitutedortho-benzyloxy (i.e. o-benzyloxy). In some embodiments, if R¹ is notunsubstituted alkyl, then R¹ is optionally 2 to 20 membered substitutedheteroalkyl.

In some embodiments, R¹ is substituted naphthyl. In some embodiments, R¹is substituted or unsubstituted acenaphthenyl. In some embodiments, R¹is substituted or unsubstituted thiophenyl-phenyl. In some embodiments,R¹ is substituted or unsubstituted naphthyl, or substituted thiophenyl,and R² is hydrogen or methyl.

In some embodiments, R² is a substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl.

R² may be selected from a substituted or unsubstituted C₁-C₂₀ alkyl,substituted or unsubstituted 2 to 20 membered heteroalkyl, substitutedor unsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted 4 to 8membered heterocycloalkyl, substituted or unsubstituted aryl, andsubstituted or unsubstituted heteroaryl. In some embodiment, R² is notconnected to the remainder of the molecule via nitrogen (i.e. anitrogen-carbon bond). In some embodiments, R² is connected to theremainder of the molecule via a carbon-carbon bond.

Alternatively, R² is unsubstituted C₁-C₂₀ alkyl; unsubstituted 2 to 20membered heteroalkyl; unsubstituted C₃-C₈ cycloalkyl; unsubstituted 4 to8 membered heterocycloalkyl; unsubstituted aryl; unsubstitutedheteroaryl; or C₁-C₂₀ alkyl, 2 to 20 membered heteroalkyl, C₃-C₈cycloalkyl, 4 to 8 membered heterocycloalkyl, aryl, or heteroaryl, thatis substituted with a substituent selected from a halogen, hydroxyl,sulfhydryl, cyano, oxy, nitro, unsubstituted C₁-C₁₀ alkyl, unsubstituted2 to 10 membered heteroalkyl, unsubstituted C₃-C₈ cycloalkyl,unsubstituted 4 to 8 membered heterocycloalkyl, unsubstituted aryl, andunsubstituted heteroaryl.

In some embodiments, R² is an unsubstituted C₁-C₁₀ alkyl; unsubstituted2 to 10 membered heteroalkyl; unsubstituted C₅ cycloalkyl; unsubstitutedphenyl; unsubstituted benzyl; or C₁-C₁₀ alkyl, 2 to 10 memberedheteroalkyl, C₅ cycloalkyl, phenyl, or benzyl, substituted with asubstituent selected from a halogen, hydroxyl, oxy, sulfhydryl, cyano,nitro, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10 memberedheteroalkyl, unsubstituted C₃-C₈ cycloalkyl, unsubstituted 4 to 8membered heterocycloalkyl, unsubstituted aryl, and unsubstitutedheteroaryl.

In other embodiments, R² is unsubstituted C₁-C₁₀ alkyl; unsubstituted 2to 10 membered heteroalkyl; unsubstituted C₅ cycloalkyl; unsubstitutedphenyl; unsubstituted benzyl; or C₁-C₁₀ alkyl or 2 to 10 memberedheteroalkyl, that is substituted with a substituent selected from ahalogen, hydroxyl, and oxy.

Alternatively, R² is a hydrogen, unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cyclokalkyl, unsubstituted aryl,unsubstituted arylalkyl, or unsubstituted alkylesteryl. R² may also bean unsubstituted C₁-C₂₀ alkyl. In some embodiments, R² is anunsubstituted C₁-C₁₀ alkyl. In other embodiments, R² is tertiary butyl.

In some embodiments, R² is R¹⁰-substituted C₁-C₂₀ alkyl, 2 to 20membered substituted or unsubstituted heteroalkyl, W¹-substituted C₃-C₈cycloalkyl, R¹¹-substituted C₃-C₈ heterocycloalkyl, R¹²-substitutedheteroaryl, or Rn-substituted aryl. R¹⁰ is oxo, —OH, halogen, —CF₃,—NH₂, 2 to 20 membered substituted or unsubstituted heteroalkyl (e.g.substituted or unsubstituted alkoxy), 3 to 7 membered substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl,or R¹³-substituted aryl. R¹¹ is oxo, —OH, halogen, —CF₃, —NH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R¹² is —OH, halogen, —CF₃,—NH₂, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl. R¹³ is —OH, —NH₂, substitutedor unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted heteroaryl, orsubstituted or unsubstituted aryl. In some related embodiments, if R³and R⁴ are hydrogen and R¹ is 4-methylphenyl, then R² is not2-ethylacetyl. In other related embodiments, if R³ and R⁴ are hydrogenand R¹ is methylphenyl or ethylphenyl, then R² is not alkylacetyl.

In certain embodiments, R³ and R⁴ are independently selected from ahydrogen, substituted or unsubstituted alkyl, and substituted orunsubstituted heteroalkyl. In some embodiments, R³ and R⁴ are,independently, a hydrogen, substituted or unsubstituted C₁-C₁₀ alkyl, orsubstituted or unsubstituted 2 to 10 membered heteroalkyl. R³ and R⁴ mayalso be independently selected from a hydrogen, unsubstituted C₁-C₁₀alkyl, and unsubstituted 2 to 10 membered heteroalkyl. Alternatively, R³and R⁴ are, independently, a hydrogen or methyl. In some embodiments, R³and R⁴ are hydrogen.

In some embodiments, R³ and R⁴ are, independently, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. In some embodiments, R³ and R⁴ areunsubstituted alkyl.

In some embodiments, R¹ is phenyl substituted with halogen, methyl, —OH,—CF₃, —OCH₃, or —NH₂; R² is C₁-C₁₀ unsubstituted alkyl; R³ and R⁴ areindependently hydrogen or methyl; and L¹ is methylene. R¹ may also bephenyl substituted with halogen (e.g. chlorine). In some embodiments, R¹is ortho-chlorophenyl. R¹ may also be phenyl substituted with halogen,—OH, —CF₃, —OCH₃, or —NH₂. In some embodiments, R³ and R⁴ are hydrogen.In some embodiments, R³ is hydrogen and R⁴ is methyl.

L¹ may be a bond, unsubstituted C₁-C₁₀ alkylene, or C₁-C₁₀ alkylenesubstituted with unsubstituted alkyl, unsubstituted heteroalkyl,unsubstituted cycloalkyl, unsubstituted heteroalkyl, unsubstituted aryl,or unsubstituted heteroaryl. In some embodiments, L¹ is methylene.

L¹ may also be a bond, substituted or unsubstituted alkylene, orsubstituted or unsubstituted heteroalkylene, wherein if R¹ is asubstituted phenyl, substituted or unsubstituted benzodioxolanyl, orsubstituted or unsubstituted cyclopentyl, then L¹ is substituted orunsubstituted alkylene, or substituted or unsubstituted heteroalkylene.

In some embodiments, L¹ is a bond or substituted or unsubstitutedalkylene, wherein if R¹ is a substituted phenyl, substituted orunsubstituted benzodioxolanyl, or unsubstituted cyclopentyl, then L¹ issubstituted or unsubstituted alkylene.

In other embodiments, L¹ is a bond or unsubstituted C₁-C₂₀ alkylene,wherein if R¹ is a phenyl, benzodioxolanyl, or cyclopentyl, then L¹ isan unsubstituted C₁-C₂₀ alkylene. In other embodiments, L¹ is a bond orunsubstituted C₁-C₂₀ alkylene, wherein if R¹ is a phenyl,benzodioxolanyl, or cyclopentyl, then L¹ is an unsubstituted C₂-C₂₀alkylene. In other embodiments, L¹ is a bond or unsubstituted C₁-C₂₀alkylene, wherein if R¹ is a phenyl, benzodioxolanyl, or cyclopentyl,then L¹ is an unsubstituted C₃-C₂₀ alkylene.

Alternatively, L¹ is a bond or unsubstituted C₁-C₁₀ alkylene, wherein ifR¹ is a phenyl, benzodioxolanyl, or cyclopentyl, then L¹ is anunsubstituted C₁-C₁₀ alkylene.

L¹ may also be a bond or unsubstituted C₁-C₅ alkylene, wherein if R¹ isa phenyl, benzodioxolanyl, or cyclopentyl, then L¹ is an unsubstitutedC₁-C₅ alkylene. Alternatively, L¹ may be a bond or methylene, wherein ifR¹ is a phenyl, benzodioxolanyl, or cyclopentyl, then L¹ is a methylene.

In some embodiments, L¹ is substituted or unsubstituted[1,1]cycloalkylene, such as substituted or unsubstituted[1,1]-cyclopropylene. In some embodiments, L¹ is unsubstituted[1,1]cycloalkylene, such as unsubstituted [1,1]-cyclopropylene.

In an exemplary embodiment, R⁵, R⁶, R⁷, R⁸ and R⁹ of Formula (II),above, are independently selected from a hydrogen; halogen; hydroxyl;sulfhydryl; cyano; nitro; substituted or unsubstituted alkyl;substituted or unsubstituted heteroalkyl; substituted or unsubstitutedcycloalkyl; substituted or unsubstituted heterocycloalkyl; substitutedor unsubstituted aryl; and substituted or unsubstituted heteroaryl.

In some embodiments, R⁵, R⁷, and R⁹ are independently selected from ahydrogen; halogen; hydroxyl; sulfhydryl; cyano; nitro; substituted orunsubstituted alkyl; substituted or unsubstituted heteroalkyl;substituted or unsubstituted cycloalkyl; substituted or unsubstitutedheterocycloalkyl; substituted or unsubstituted aryl; and substituted orunsubstituted heteroaryl. R⁶ and R⁸ are independently selected from ahydrogen, halogen, hydroxyl, sulfhydryl, cyano, nitro, substituted orunsubstituted alkyl, unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and heteroalkyl substituted with a substituted aryl. Incertain embodiments, R⁵, R⁶, R⁸, and R⁹ are not simultaneously hydrogen.

In other embodiments, R⁵, R⁷, and R⁹ are, independently, hydrogen,halogen, hydroxyl, sulfhydryl, cyano, nitro, substituted orunsubstituted C₁-C₂₀ alkyl, substituted or unsubstituted 2 to 20membered heteroalkyl, substituted or unsubstituted C₃-C₈ cycloalkyl,substituted or unsubstituted 4 to 8 membered heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

Alternatively, R⁵, R⁷, and R⁹ are, independently, hydrogen; halogen;hydroxyl; sulfhydryl; cyano; nitro; unsubstituted C₁-C₁₀ alkyl;unsubstituted 2 to 10 membered heteroalkyl; unsubstituted C₃-C₈cycloalkyl; unsubstituted 4 to 8 membered heterocycloalkyl;unsubstituted aryl; unsubstituted heteroaryl; or C₁-C₁₀ alkyl, 2 to 20membered heteroalkyl, C₃-C₈ cycloalkyl, 4 to 8 memberedheterocycloalkyl, aryl, or heteroaryl, that is substituted with asubstituent selected from a halogen, hydroxyl, sulfhydryl, cyano, nitro,unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl,unsubstituted C₃-C₈ cycloalkyl, unsubstituted 4 to 8 memberedheterocycloalkyl, unsubstituted heteroaryl, unsubstituted aryl, and arylsubstituted with a halogen or unsubstituted heteroaryl.

R⁵, R⁷, and R⁹ may also be, independently, hydrogen; halogen; hydroxyl;unsubstituted C₁-C₁₀ alkyl; unsubstituted 2 to 10 membered heteroalkyl;unsubstituted C₅-C₈ cycloalkyl; unsubstituted 5 to 8 memberedheterocycloalkyl; unsubstituted aryl; unsubstituted heteroaryl; orC₁-C₁₀ alkyl or 2 to 10 membered heteroalkyl, that is substituted with asubstituent selected from an unsubstituted aryl, unsubstitutedheteroaryl, and aryl substituted with a halogen.

In another embodiment, R⁵, R⁷, and R⁹ are independently selected from ahydrogen, halogen, unsubstituted C₁-C₁₀ alkyl, and unsubstituted 2 to 10membered heteroalkyl. R⁵, R⁷, and R⁹ may also independently be ahydrogen, halogen, methyl, or methoxy.

In another embodiment, R⁶ and R⁸ are, independently, hydrogen, halogen,hydroxyl, sulfhydryl, cyano, nitro, substituted or unsubstituted C₁-C₂₀alkyl, unsubstituted 2 to 20 membered heteroalkyl, substituted orunsubstituted C₃-C₈ cycloalkyl, substituted or unsubstituted 4 to 8membered heterocycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or 2 to 20 membered heteroalkylsubstituted with a substituted aryl.

In some embodiments, R⁶ and R⁸ are, independently, hydrogen; halogen;hydroxyl; sulfhydryl; cyano; nitro; unsubstituted C₁-C₁₀ alkyl;unsubstituted 2 to 10 membered heteroalkyl; unsubstituted C₃-C₈cycloalkyl; unsubstituted 4 to 8 membered heterocycloalkyl;unsubstituted aryl; unsubstituted heteroaryl; 2 to 10 memberedheteroalkyl substituted with a substituted aryl; or C₁-C₁₀ alkyl, C₃-C₈cycloalkyl, 4 to 8 membered heterocycloalkyl, aryl, or heteroaryl,substituted with a substituent selected from a halogen, hydroxyl,sulfhydryl, cyano, nitro, unsubstituted C₁-C₁₀ alkyl, unsubstituted 2 to10 membered heteroalkyl, unsubstituted C₃-C₈ cycloalkyl, unsubstituted 4to 8 membered heterocycloalkyl, unsubstituted aryl, and unsubstitutedheteroaryl.

In other embodiments, R⁶ and R⁸ are, independently, hydrogen; halogen;hydroxyl; unsubstituted C₁-C₁₀ alkyl; unsubstituted 2 to 10 memberedheteroalkyl; unsubstituted C₃-C₈ cycloalkyl; unsubstituted 4 to 8membered heterocycloalkyl; unsubstituted aryl; unsubstituted heteroaryl;2 to 10 membered heteroalkyl substituted with a haloaryl; or C₁-C₁₀alkyl substituted with a substituent selected from an unsubstitutedaryl, haloaryl, or unsubstituted heteroaryl.

R⁶ and R⁸ may also be selected from a hydrogen, halogen, unsubstitutedC₁-C₁₀ alkyl, unsubstituted 2 to 10 membered heteroalkyl, or 2 to 5membered heteroalkyl substituted with a haloaryl.

Alternatively, R⁶ and R⁸ are independently selected from a hydrogen,halogen, methyl, methoxy, and methoxy substituted with a haloaryl.

In some embodiments, each substituted group described above in thecompounds of Formulae (I) and/or (II) is substituted with at least onesubstituent group. Fore example, in some embodiments, each substitutedalkyl, substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted cycloalkylene, and/or substituted heteroalkylene,described above in the compounds of Formulae (I) and/or (II) aresubstituted with at least one substituent group. In other embodiments,at least one or all of these groups are substituted with at least onesize-limited substituent group. Alternatively, at least one or all ofthese groups are substituted with at least one lower substituent group.

In other embodiments of the compounds of Formulae (I) and/or (II), eachsubstituted or unsubstituted alkyl is a substituted or unsubstitutedC₁-C₂₀ alkyl, each substituted or unsubstituted heteroalkyl is asubstituted or unsubstituted 2 to 20 membered heteroalkyl, eachsubstituted or unsubstituted cycloalkyl is a substituted orunsubstituted C₃-C₈ cycloalkyl, each substituted or unsubstitutedheterocycloalkyl is a substituted or unsubstituted 4 to 8 memberedheterocycloalkyl, each substituted or unsubstituted alkylene is asubstituted or unsubstituted C₁-C₂₀ alkylene, each substituted orunsubstituted cycloalkylene is a C₃-C₈ cycloalkylene, and/or eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene.

Alternatively, each substituted or unsubstituted alkyl is a substitutedor unsubstituted C₁-C₈ alkyl, each substituted or unsubstitutedheteroalkyl is a substituted or unsubstituted 2 to 8 memberedheteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₅-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7membered heterocycloalkyl, each substituted or unsubstituted alkylene isa substituted or unsubstituted C₁-C₈ alkylene, each substituted orunsubstituted cycloalkylene is a C₃-C₆ cycloalkylene, and/or eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene.

In some embodiments, the compounds of the invention are the subset ofcompounds set forth in Table 1 below, with the exception of compound 13and/or compound 57. Although compound 57 does not fall within the scopeof Formula (I), in some embodiments compound 57 of Table 1 is useful inthe methods of the present invention. In some embodiments, the compoundsof the invention are the subset of compounds set forth in Table 1 below,with the exception of compound 57. In some embodiments, the compounds ofthe invention are the subset of compounds in Table 1 that are set forthas “active.” In some embodiments, the compounds of the invention do notinclude those compounds set forth in Table 2 below. One skilled in theart, using the methods described herein and/or methods well known in theart, can easily determine which compounds are useful asanti-inflammatory compounds (see e.g. section III below)

II. Exemplary Synthesis

The compounds of the invention are synthesized by an appropriatecombination of generally well known synthetic methods. Techniques usefulin synthesizing the compounds of the invention are both readily apparentand accessible to those of skill in the relevant art in view of thedisclosure herein. The discussion below is offered to illustrate certainof the diverse methods available for use in assembling the compounds ofthe invention. However, the discussion is not intended to define thescope of reactions or reaction sequences that are useful in preparingthe compounds of the present invention.

In Scheme 1, R¹ and L¹ are as defined above. In step (i), carbonylcompound I is reacted with NaH and malononitrile in THF, followed by(ii) treatment with NaHCO₃, and dimethyl sulfate in dioxane/H₂O to formthe corresponding alkene 2. Cyclization of 2 is accomplished byrefluxing in the presence of triethylamine and tert-butylhydrazinehydrochloride in ethanol to from the pyrazole 3. Finally, 3 is reactedwith formamide to yield the pyrazolopyrimidine 4.

The amine substituent of 4 may be derivatized by reacting with theappropriate acyl chloride (R′—C(O)—Cl) in the presence of pyridine (v)(Scheme 2). The resulting amide may be reduced by refluxing with LiAlH₄in dry tetrahydrofuran to form 6. In scheme 2, R′ represents substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl. In some embodiments, the —CH₂—R′ moiety is R³,which is defined above.

For a more detailed discussion of useful synthesis procedures, seeBishop et al., J. Am. Chem. Soc. 121, 627-631 (1999) and Bishop et al.,Current Biology, 8: 257-266 (1998).

III. Methods of Identifying an Anti-Inflammatory Pyrazolopyrimidine

In another aspect, the present invention provides a method foridentifying anti-inflammatory pyrazolopyrimidine compounds. The methodincludes contacting a candidate anti-inflammatory pyrazolopyrimidinecompound with a cell culture assay. The cell culture assay may includemore than one type of cell, also referred to herein as a cell cultureassay combination. The cell culture assay combination may include, forexample and without limitation, human endothelial cells in aninflammatory state with or without peripheral blood mononuclear cells.In one type of cell culture assay, changes in the expression of at leasttwo different gene products relevant to an inflammatory state aredetected. The change in the expression of the at least two differentgene products is compared to the expression of those gene products inthe absence of the candidate anti-inflammatory pyrazolopyrimidinecompound, and if the candidate compound alters the expression of one ormore of such gene products in a manner consistent with anti-inflammatoryaction (for example, by decreasing the expression of a gene product thatinduces an inflammatory state or is characteristic of an inflammatorystate or by increasing the expression of a gene product that inhibitsinflammation), then the candidate compound is thereby identified as ananti-inflammatory pyrazolopyrimidine compound. Cell culture assays andmethods for analyzing the data obtained by them useful in this aspect ofthe invention are described in PCT publication Nos. 05/023987;04/094992; 04/094609; 04/022711; 03/023753; and 01/067103; and U.S. Pat.Nos. 6,656,695; and 6,763,307, each of which is incorporated herein byreference.

In some embodiments, the gene products analyzed are the products of agene selected from E-selectin, ICAM-1, VCAM-1, CXLC9/MIG, IL-6,CXCL8/IL-8, HLA-DR, CCL2/MCP-1, P-selectin, VEGFR2, CD87/uPAR, CD55,CCL26/Eotaxin-3, CD14, CD40, CD69, CD31, CD38, CD142/TF, IL-1α, M-CSF,CD141/TM, Endothelin-1, LDLR, CXCL10/IP-10, CD3, and IL-2. Additionalgene products that may be analyzed in such assays are discussed indetail, for example, in the above cited PCT patent publications andissued US patents, each of which has been incorporated by reference intheir entirety for all purposes. Other exemplary targets and pathwaysthat may be detected in such cell culture assays are illustrated inFIGS. 1A and 1B below.

The inflammatory state of the cells in the cell culture assay may beachieved by adding to the culture one or more inflammatory factorsselected from TNF-α, TNF-β, IL-1, IL-2, IL-4, IL-12, IL-13,Staphylococcal Enterotoxin B (SEB), Staphylococcal Enterotoxin E (SEE),toxic shock syndrome toxin (TSST), lipopolysaccharide (LPS), anti-CD3antibody, anti-T cell receptor antibody, histamine and IFN-γ. Theinflammatory factors are typically added in an amount and for a timesufficient to induce said inflammatory state. In some embodiments, atleast one, two or three inflammatory factors are added. Additionalinflammatory factors are discussed in detail, for example, in the abovecited PCT patent publications and issued US patents.

In certain embodiments, activities of a candidate pyrazolopyrimidinecompound are compared to a known anti-inflammatory compound.Pyrazolopyrimidine compounds with novel activity profiles or withactivity profiles similar to other anti-inflammatory compounds maythereby be detected.

In an exemplary embodiment, a Biologically Multiplexed ActivityProfiling system, known under the mark BioMAP™ system, is employed.Useful BioMAP systems are discussed in detail, for example, in the abovecited PCT patent publications and issued US patents and in Kunkel etal., ASSAY and Drug Development Technologies, 2: 431-441 (2004),incorporated herein by reference.

Methods for selecting cells and culture conditions for screeningcandidate anti-inflammatory pyrazolopyrimidines, identification of anoptimized set of discrete parameters to be measured, the use of BioMAPanalysis for rapid identification and characterization of candidates,and the like, are within the skill of those having knowledge in thebiological and/or biochemical arts in view of the disclosure herein andthat of the above cited PCT patent publications and issued US patents.In some embodiments, a large number of cellular pathways, and the rapididentification of compounds that cause cellular responses, aresimultaneously screened.

Agents are screened for biological activity by adding an agent to bescreened to at least one and usually a plurality of assay combinationsto form a panel of assay combinations, usually in conjunction with assaycombinations lacking agent. The change in parameter readout in responseto agent is measured, desirably normalized, and the resulting BioMAPreadout may then be evaluated by comparison to reference BioMAPreadouts. The reference BioMAP readouts may include basal readouts inthe presence and absence of the factors, BioMAP readouts obtained withother agents, which may or may not include known inhibitors of knownpathways. Agents of interest for analysis include any biologicallyactive molecule with the capability of modulating, directly orindirectly, the phenotype of interest of a cell of interest.

Various methods can be utilized for quantifying the presence of theselected markers. For measuring the amount of a molecule that ispresent, a convenient method is to label a molecule with a detectablemoiety, which may be fluorescent, luminescent, radioactive,enzymatically active, etc., particularly a molecule specific for bindingto the parameter with high affinity Fluorescent moieties are readilyavailable for labeling virtually any biomolecule, structure, or celltype. Immunofluorescent moieties can be directed to bind not only tospecific proteins but also specific conformations, cleavage products, orsite modifications like phosphorylation. Individual peptides andproteins can be engineered to autofluoresce, e.g. by expressing them asgreen fluorescent protein chimeras inside cells (for a review see Joneset al. (1999) Trends Biotechnol., 17(12):477-81). Thus, antibodies canbe genetically modified to provide a fluorescent dye as part of theirstructure. Applicable methods are generally known in art, and arediscussed in detail, for example, in the above cited PCT patentpublications and issued US patents.

In certain embodiments, human umbilical vein endothelial cells (HUVEC)and/or peripheral blood mononuclear cells (PBMC) are contacted withdifferent combinations of cytokines to induce an inflammatory response.Exemplary cytokine combinations are illustrated in FIG. 1B. Theinflammatory response is assessed by detecting the presence of the geneproducts shown in FIG. 1B.

IV. Methods for Treating or Preventing a Disorder Characterized byInflammation

In another aspect, the present invention provides a method for treatingor preventing a disorder characterized by inflammation, said methodincluding administering to a subject a therapeutically effective amountof a compound of the present invention.

In certain embodiments, the inflammatory process that occurs in responseto or is an underlying cause of infection, trauma, autoimmune disease,cardiovascular disease, neoplasia, hyperplasia, addiction, infection,obesity, cellular degeneration, apoptosis, or senescence, ordifferentiation.

The disorder may be selected from the group consisting of vasculitis,multiple sclerosis, diabetes, inflammatory bowel disease (e.g. Crohn'sdisease and ulcerative colitis), psoriasis, arthritis (e.g. rheumatoidarthritis, spondyloarthopathies, gouty arthritis, osteoarthritis, andjuvenile arthritis), asthma, stroke, atherosclerosis (includingatherosclerotic plaque rupture), restenosis, and lupus (includingsystemic lupus erythematosus).

Other disorders associated with abnormal inflammation include, forexample, headaches (e.g. migraine headaches), bronchitis, menstrualcramps, tendonitis, gastritis, vascular diseases, uveitis, Sjogren'sdisease, sclerodoma, nephrotic syndrome, swelling occurring afterinjury, myocardial ischemia, fever (including rheumatic fever and feverassociated with influenza and other viral infections), common cold,dysmenorrhea, emphysema, acute respiratory distress syndrome, chronicobstructive pulmonary disease, Alzheimer's disease, organ transplanttoxicity, cachexia, allergic reactions, allergic contacthypersensitivity, cancer (such as solid tumor cancer including coloncancer, breast cancer, lung cancer and prostrate cancer; hematopoieticmalignancies including leukemias and lymphomas; Hodgkin's disease;aplastic anemia, skin cancer and familiar adenomatous polyposis), tissueulceration, peptic ulcers, regional enteritis, ulcerative colitis,diverticulitis, recurrent gastrointestinal lesion, gastrointestinalbleeding, coagulation, synovitis, gout, ankylosing spondylitis,restenosis, periodontal disease, epidermolysis bullosa, osteoporosis,loosening of artificial joint implants, aortic aneurysm (includingabdominal aortic aneurysm and brain aortic aneurysm), periarteritisnodosa, congestive heart failure, myocardial infarction, cerebralischemia, head trauma, spinal cord injury, neuralgia, neuro-degenerativedisorders (acute and chronic), autoimmune disorders, Huntington'sdisease, Parkinson's disease, migraine, depression, peripheralneuropathy, pain (including low back and neck pain, headache andtoothache), gingivitis, cerebral amyloid angiopathy, nootropic orcognition enhancement, amyotrophic lateral sclerosis, multiplesclerosis, ocular angiogenesis, corneal injury, macular degeneration,conjunctivitis, abnormal wound healing, muscle or joint sprains orstrains, tendonitis, skin disorders (such as psoriasis, eczema,scleroderma and dermatitis), myasthenia gravis, polymyositis, myositis,bursitis, burns, diabetes (including types I and II diabetes, diabeticretinopathy, neuropathy and nephropathy, insulin dependent diabetesmellitus (IDDM)), tumor invasion, tumor growth, tumor metastasis,corneal scarring, scleritis, immunodeficiency diseases (such as AIDS inhumans and FLV, FIV in cats), sepsis, premature labor,hypoprothrombinemia, hemophilia, thyroiditis, sarcoidosis, Behcet'ssyndrome, hypersensitivity, kidney disease, Rickettsial infections (suchas Lyme disease, Erlichiosis), Protozoan diseases (such as malaria,giardia, coccidia), reproductive disorders (preferably in livestock) andseptic shock.

Treatment may be conducted in a mammal (e.g. a human, cat, dog, orlivestock), and the treatment method comprises administering atherapeutically effective amount of a compound of Formula (I) or apharmaceutically acceptable salt thereof in combination with apharmaceutically acceptable carrier.

V. Anti-Inflammatory Activity

As discussed above, the present invention provides anti-inflammatorycompounds useful in the treatment of diseases and conditions in whichinflammation is involved in disease progression or the manifestation ofsymptoms of the disease or condition.

In one embodiment, the compounds of the invention are the subset ofcompounds of Formula (I) that are T cell activation inhibitors. T-cellactivation inhibitors are those compounds that strongly inhibit T cellactivation in a cell-based assay (e.g. a BioMAP assay as describedabove, where, for example, a superantigen is used to stimulate T cells).The phrase “strongly inhibit T cell activation,” as used herein, refersto compounds that inhibit the expression of at least one gene productselected from E-selectin, ICAM-1, VCAM-1, CXLC9/MIG, CXCL8/IL-8,CCL2/MCP-1, CD87/uPAR, CCL26/Eotaxin-3, CD40, CD69, CD38, CD142/TF,CXCL10/IP-10, or IL-2 by at least 50% in a cell-based assay. T-cellactivation inhibitors may be especially useful in treating theinflammation underlying arthritis, psoriasis, vasculitis, multiplesclerosis, IBD, asthma, atherosclerosis, or other inflammatory disease.Illustrative pyrazolopyrimidine T cell activation inhibitor compounds ofthis embodiment include compounds 10, 13, 15, 17, 41, 48, and 54 inTable 1 below.

In another embodiment, the compounds of the invention are the subset ofcompounds of Formula (I0 that are monocyte activation and recruitmentinhibitors. Monocyte activation inhibitors weakly inhibit T cellactivation, strongly inhibit monocyte activation, and strongly inhibitMCP-1 and IL-8 production from multiple cell types including but notlimited to endothelial cells, fibroblasts, and smooth muscle cells. Thephrase “weakly inhibit T cell activation,” as used herein, refers tocompounds that inhibit the expression of at least one gene productselected from E-selectin, ICAM-1, VCAM-1, CXLC9/MIG, CXCL8/IL-8,CCL2/MCP-1, CD87/uPAR, CCL26/Eotaxin-3, CD40, CD69, CD38, CD142/TF,CXCL10/IP-10, or IL-2 by less than 50% in a cell-based assay. The phrase“strongly inhibit monocyte activation,” as used herein, refers tocompounds that inhibit the expression of at least one gene productselected from E-selectin, ICAM-1, VCAM-1, CXLC9/MIG, IL-6, CXCL8/IL-8,CCL2/MCP-1, CD14, CD40, CD69, CD142/TF, IL-1α, M-CSF, CD141/TM, orCXCL10/IP-10 by at least 50% in a cell-based assay. The phrase “stronglyinhibit strongly inhibit MCP-1,” as used herein, refers to compoundsthat inhibit the expression of MCP-1 gene product by at least 50% in acell-based assay. The phrase “strongly inhibit strongly inhibit IL-8,”as used herein, refers to compounds that inhibit the expression of IL-8gene product by at least 50% in a cell-based assay. Monocyte activationinhibitors may be especially useful in treating the inflammationunderlying multiple diseases including rheumatoid arthritis, ankylosingspondylitis, psoriatic arthritis, multiple sclerosis, psoriasis,inflammatory bowel disease, atherosclerosis, diabetes, lupus nephritis,and vasculitis. Illustrative pyrazolopyrimidine monocyte activationinhibitor compounds of this embodiment include compounds 30, 31, 36, 59,62, 63, 64, 65, 72, and 76 in Table 1 below.

In another embodiment, the compounds of the invention are the subset ofcompounds of Formula (I) that are gamma interferon signaling inhibitors.Gamma interferon signaling inhibitors weakly inhibit T cell activation(see above) and monocyte activation (see above) but inhibit IFN-gammasignaling and collagen production. The phrase “weakly inhibit monocyteactivation,” as used herein, refers to compounds that inhibit theexpression of at least one gene product selected from E-selectin,ICAM-1, VCAM-1, CXLC9/MIG, IL-6, CXCL8/IL-8, CCL2/MCP-1, CD14, CD40,CD69, CD142/TF, IL-1α, M-CSF, CD141/TM, or CXCL10/IP-10 by less than 50%in a cell-based assay. Gamma interferon signaling inhibitors may beespecially useful in treating the inflammation underlying multiplediseases including rheumatoid arthritis, systemic sclerosis, COPD,asthma, atherosclerosis, cirrhosis, pulmonary fibrosis, sarcoidosis,keloids, and renal fibrosis. Illustrative pyrazolopyrimidine interferongamma signaling inhibitor compounds of this embodiment include compounds7, 8, 9, 11, 12, and 19 in Table 1 below.

VI. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticalcompositions comprising a pharmaceutically acceptable excipient and acompound of the present invention, such as a compound within the scopeof Formula (I) provided above.

The compounds of the present invention can be prepared and administeredin a wide variety of oral, parenteral and topical dosage forms. Oralpreparations include tablets, pills, powder, dragees, capsules, liquids,lozenges, gels, syrups, slurries, suspensions, etc., suitable foringestion by the patient. The compounds of the present invention canalso be administered by injection, that is, intravenously,intramuscularly, intracutaneously, subcutaneously, intraduodenally, orintraperitoneally. Also, the compounds described herein can beadministered by inhalation, for example, intranasally. Additionally, thecompounds of the present invention can be administered transdermally.The anti-inflammatory pyrazolopyrimidines of this invention can also beadministered by intraocular, intravaginal, and intrarectal routesincluding via suppositories, insufflation, powders and aerosolformulations (for examples of steroid inhalants, see Rohatagi, J Clin.Pharmacol. 35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol.75:107-111, 1995). Accordingly, the present invention also providespharmaceutical compositions comprising a pharmaceutically acceptablecarrier or excipient and either a compound of Formula (I), or apharmaceutically acceptable salt of a compound of Formula (I).

The compounds of the present invention may also be administered using ananti-inflammatory pyrazolopyrimidine coated stent. The stent may beinserted into a vessel, acting as a scaffold to provide structuralsupport for the vessel thereby holding the vessel open and improvingblood flow through the vessel. An anti-inflammatory pyrazolopyrimidinecoated stent refers to a stent that has been coated withanti-inflammatory pyrazolopyrimidine and optionally additional agents,such as an appropriate pharmaceutical excipient. The anti-inflammatorypyrazolopyrimidine coated stent typically allows the anti-inflammatorypyrazolopyrimidine to be released over time into surrounding tissue. Oneskilled in the art understands that the stent may be adsorbed to,impregnated with, covalently attached to, or ionically bonded to theanti-inflammatory pyrazolopyrimidine.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispersible granules. Asolid carrier can be one or more substances, which may also act asdiluents, flavoring agents, binders, preservatives, tabletdisintegrating agents, or an encapsulating material. Details ontechniques for formulation and administration are well described in thescientific and patent literature, see, e.g., the latest edition ofRemington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.(“Remington's”).

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided active component. In tablets, active componentis mixed with a carrier having the necessary binding properties insuitable proportions and compacted in the shape and size desired.

The powders and tablets preferably contain from 5% or 10% to 70% ofactive compound. Suitable carriers are magnesium carbonate, magnesiumstearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin,tragacanth, methylcellulose, sodium carboxymethylcellulose, a lowmelting wax, cocoa butter, and the like. The term “preparation” isintended to include the formulation of active compound withencapsulating material as a carrier providing a capsule in which activecomponent with or without other carriers, is surrounded by a carrier,which is thus in association with it. Similarly, cachets and lozengesare included within the pharmaceutical formulations of the invention.Tablets, powders, capsules, pills, cachets, and lozenges can be used assolid dosage forms suitable for oral administration.

Suitable solid excipients are carbohydrate or protein fillers andinclude, but are not limited to, sugars, including lactose, sucrose,mannitol, or sorbitol; starch from corn, wheat, rice, potato, or otherplants; cellulose such as methyl cellulose,hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; andgums including arabic and tragacanth; as well as proteins such asgelatin and collagen. If desired, disintegrating or solubilizing agentsmay be added, such as the cross-linked polyvinyl pyrrolidone, agar,alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores are provided with suitable coatings such as concentratedsugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound (i.e., dosage). Pharmaceutical preparations of theinvention can also be used orally using, for example, push-fit capsulesmade of gelatin, as well as soft, sealed capsules made of gelatin and acoating such as glycerol or sorbitol. Push-fit capsules can contain ananti-inflammatory pyrazolopyrimidine mixed with a filler or binders suchas lactose or starches, lubricants such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the anti-inflammatorypyrazolopyrimidines may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycol withor without stabilizers.

For preparing suppositories, a low melting wax, such as a mixture offatty acid glycerides or cocoa butter, is first melted and activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogeneous mixture is then poured into convenient sized molds, allowedto cool, and thereby to solidify.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water/propylene glycol solutions. For parenteralinjection, liquid preparations can be formulated in solution in aqueouspolyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolvingactive component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).Aqueous suspension can also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Also included are solid form preparations, which are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to activecomponent, colorants, flavors, stabilizers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilizing agents, andthe like.

Oil suspensions can be formulated by suspending an anti-inflammatorypyrazolopyrimidine in a vegetable oil, such as arachis oil, olive oil,sesame oil or coconut oil, or in a mineral oil such as liquid paraffin;or a mixture of these. The oil suspensions can contain a thickeningagent, such as beeswax, hard paraffin or cetyl alcohol. Sweeteningagents can be added to provide a palatable oral preparation, such asglycerol, sorbitol or sucrose. These formulations can be preserved byaddition of an antioxidant such as ascorbic acid. As an example of aninjectable oil vehicle, see Minto, J Pharmacol. Exp. Ther. 281:93-102,1997. The pharmaceutical formulations of the invention can also be inthe form of oil-in-water emulsions. The oily phase can be a vegetableoil or a mineral oil, described above, or a mixture of these. Suitableemulsifying agents include naturally-occurring gums, such as gum acaciaand gum tragacanth, naturally occurring phosphatides, such as soybeanlecithin, esters or partial esters derived from fatty acids and hexitolanhydrides, such as sorbitan mono-oleate, and condensation products ofthese partial esters with ethylene oxide, such as polyoxyethylenesorbitan mono-oleate. The emulsion can also contain sweetening agentsand flavoring agents, as in the formulation of syrups and elixirs. Suchformulations can also contain a demulcent, a preservative, or a coloringagent.

The anti-inflammatory pyrazolopyrimidines of the invention can bedelivered transdermally, by a topical route, formulated as applicatorsticks, solutions, suspensions, emulsions, gels, creams, ointments,pastes, jellies, paints, powders, and aerosols.

The anti-inflammatory pyrazolopyrimidines of the invention can also bedelivered as microspheres for slow release in the body. For example,microspheres can be administered via intradermal injection ofdrug-containing microspheres, which slowly release subcutaneously (seeRao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable andinjectable gel formulations (see, e.g., Gao Pharm. Res. 12:857-863,1995); or, as microspheres for oral administration (see, e.g., Eyles, JPharm. Pharmacol. 49:669-674, 1997). Both transdermal and intradermalroutes can in some instances afford constant or near constant rates ofdelivery of the active agents for weeks or months.

The anti-inflammatory pyrazolopyrimidine pharmaceutical formulations ofthe invention can be provided as a salt and can be formed with manyacids, including but not limited to hydrochloric, sulfuric, acetic,lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble inaqueous or other protonic solvents that are the corresponding free baseforms. In other cases, the preparation may be a lyophilized powder in 1mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

In another embodiment, the anti-inflammatory pyrazolopyrimidineformulations of the invention are used for parenteral administration,such as intravenous (IV) administration or administration into a bodycavity or lumen of an organ. The formulations for administration willcommonly comprise a solution of the anti-inflammatory pyrazolopyrimidinedissolved in a pharmaceutically acceptable carrier. Among acceptablevehicles and solvents that can be employed are water and Ringer'ssolution, an isotonic sodium chloride. In addition, sterile fixed oilscan conventionally be employed as a solvent or suspending medium. Forthis purpose, any bland fixed oil can be employed including syntheticmono- or diglycerides. In addition, fatty acids such as oleic acid canlikewise be used in the preparation of injectables. These solutions aresterile and generally free of undesirable matter. These formulations maybe sterilized by conventional, well known (for other compounds)sterilization techniques. The formulations may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents, e.g., sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate and the like. The concentration ofanti-inflammatory pyrazolopyrimidine in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated according to the known artusing those suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation can also be a sterile injectablesolution or suspension in a nontoxic parenterally-acceptable diluent orsolvent, such as a solution of 1,3-butanediol.

In another embodiment, the anti-inflammatory pyrazolopyrimidineformulations of the invention can be delivered by the use of liposomeswhich fuse with the cellular membrane or are endocytosed, i.e., byemploying ligands attached to the liposome, or attached directly to theoligonucleotide, that bind to surface membrane protein receptors of thecell resulting in endocytosis. By using liposomes, particularly wherethe liposome surface carries ligands specific for target cells, or areotherwise preferentially directed to a specific organ, one can focus thedelivery of the anti-inflammatory pyrazolopyrimidine into the targetcells in vivo. (See, e.g., Al-Muhammed, J Microencapsul. 13:293-306,1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J.Hosp. Pharm. 46:1576-1587, 1989).

The pharmaceutical preparation is preferably in unit dosage form. Insuch form the preparation is subdivided into unit doses containingappropriate quantities of active component. The unit dosage form can bea packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be appropriate number of any of these inpackaged form.

The quantity of active component in a unit dose preparation may bevaried or adjusted from 0.1 mg to 10000 mg, more typically 1.0 mg to1000 mg, most typically 10 mg to 500 mg, according to the particularapplication and the potency of active component. The composition can, ifdesired, also contain other compatible therapeutic agents.

VII. Therapeutic Combinations

One of ordinary skill in art will appreciate that the compounds of theinvention are useful in treating a diverse array of diseases. One ofordinary skill in art will also appreciate that, when using thecompounds of the invention in the treatment of a specific disease, thecompounds of the invention may be combined or otherwise co-administeredwith various existing therapeutic agents used for that disease. Forexample, for the treatment of rheumatoid arthritis, the compounds of theinvention may be combined or co-administered with agents such asTNF-αinhibitors such as anti-TNF monoclonal antibodies and TNF receptorimmunoglobulin molecules (such as Enbrel.R™), low dose methotrexate,lefunimide, hydroxychloroquine, d-penicilamine, auranofin or parenteralor oral gold.

The compounds of the invention can also be used in combination withexisting therapeutic agents for the treatment of osteoarthritis.Suitable agents to be used in combination include standard non-steroidalanti-inflammatory agents (hereinafter NSAIDs) such as piroxicam,diclofenac, propionic acids such as naproxen, flurbiprofen, fenoprofen,ketoprofen and ibuprofen, fenamates such as mefenamic acid,indomethacin, sulindac, apazone, pyrazolones such as phenylbutazone,salicylates such as aspirin, COX-2 inhibitors such as celecoxib androfecoxib, analgesics and intraarticular therapies such ascorticosteroids and hyaluronic acids such as hyalgan and synvisc.

Active ingredient (i.e. the compounds of Formula (I)) of the presentinvention may be administered in combination with inhibitors of othermediators of inflammation, comprising one or more members selected fromthe group consisting essentially of the classes of such inhibitors andexamples thereof which include, matrix metalloproteinase inhibitors,aggrecanase inhibitors, TACE inhibitors, leukotriene receptorantagonists, IL-1 processing and release inhibitors, IL-IRA, H₁-receptorantagonists; kinin-B₁- and B₁-receptor antagonists; prostaglandininhibitors such as PGD-, PGF- PGI₂-, and PGE-receptor antagonists;thromboxane A₂(TXA2-) inhibitors; 5- and 12-lipoxygenase inhibitors;leukotriene LTC₄-, LTD₄/LTE₄-, and LTB₄-inhibitors; PAF-receptorantagonists; gold in the form of an aurothio group together with varioushydrophilic groups; immunosuppressive agents, e.g., cyclosporine,azathioprine, and methotrexate; anti-inflammatory glucocorticoids;penicillamine; hydroxychloroquine; anti-gout agents, e.g., colchicine,xanthine oxidase inhibitors, e.g., allopurinol, and uricosuric agents,e.g., probenecid, sulfinpyrazone, and benzbromarone.

The compounds of the present invention may also be used in combinationwith anticancer agents such as endostatin and angiostatin or cytotoxicdrugs such as adriamycin, daunomycin, cis-platinum, etoposide, taxol,taxotere and alkaloids, such as vincristine, and antimetabolites such asmethotrexate.

The compounds of the present invention may also be used in combinationwith anti-hypertensives and other cardiovascular drugs intended tooffset the consequences of atherosclerosis, including hypertension,myocardial ischemia including angina, congestive heart failure, andmyocardial infarction, selected from vasodilators such as hydralazine,β-adrenergic receptor antagonists such as propranolol, calcium channelblockers such as nifedipine, α₂-adrenergic agonists such as clonidine,α-adrenergic receptor antagonists such as prazosin, andHMG-CoA-reductase inhibitors (anti-hypercholesterolemics) such aslovastatin or atorvastatin.

Active ingredient of the present invention may also be administered incombination with one or more antibiotic, antifungal, antiprotozoal,antiviral or similar therapeutic agents.

The compounds of the present invention may also be used in combinationwith CNS agents such as antidepressants (such as sertraline),anti-Parkinsonian drugs (such as L-dopa, requip, mirapex, MAOBinhibitors such as selegine and rasagiline, comP inhibitors such asTasmar, A-2 inhibitors, dopamine reuptake inhibitors, NMDA antagonists,nicotine agonists, dopamine agonists and inhibitors of neuronal nitricoxide synthase), and anti-Alzheimer's drugs such as donepezil, tacrine,COX-2 inhibitors, propentofylline or metrifonate.

The compounds of the present invention may also be used in combinationwith osteoporosis agents such as roloxifene, lasofoxifene, droloxifeneor fosomax and immunosuppressant agents such as FK-506 and rapamycin.

The present invention also relates to the formulation of active agentsof the present invention alone or with one or more other therapeuticagents which are to form the intended combination, including whereinsaid different drugs have varying half-lives, by creatingcontrolled-release forms of said drugs with different release timeswhich achieves relatively uniform dosing; or, in the case of non-humanpatients, a medicated feed dosage form in which said drugs used in thecombination are present together in admixture in said feed composition.There is further provided in accordance with the present inventionco-administration in which the combination of drugs is achieved by thesimultaneous administration of said drugs to be given in combination;including co-administration by means of different dosage forms androutes of administration; the use of combinations in accordance withdifferent but regular and continuous dosing schedules whereby desiredplasma levels of said drugs involved are maintained in the patient beingtreated, even though the individual drugs making up said combination arenot being administered to said patient simultaneously.

This invention also relates to a method of or a pharmaceuticalcomposition for treating inflammatory processes and diseases comprisingadministering a compound of this invention or its salt to a mammalincluding a human, cat, livestock or dog (preferably a dog), whereinsaid inflammatory processes and diseases are defined as above, and saidinhibitory compound is used in combination with one or more othertherapeutically active agents under the following conditions:

-   A.) where a joint has become seriously inflamed as well as infected    at the same time by bacteria, fungi, protozoa, and/or virus, said    inhibitory compound is administered in combination with one or more    antibiotic, antifungal, antiprotozoal, and/or antiviral therapeutic    agents;-   B.) where a multi-fold treatment of pain and inflammation is    desired, said inhibitory compound is administered in combination    with inhibitors of other mediators of inflammation, comprising one    or more members independently selected from the group consisting    essentially of:    -   (1) NSAID's;    -   (2) H₄-receptor antagonists;    -   (3) kinin-B₄- and B₄-receptor antagonists;    -   (4) prostaglandin inhibitors selected from the group consisting        of PGD-, PGF- PGI₂-, and PGE-receptor antagonists;    -   (5) thromboxane A₂ (TXA₂-) inhibitors;    -   (6) 5-, 12- and 15-lipoxygenase inhibitors;    -   (7) leukotriene LTC₄-, LTD₄/LTE₄-, and LTB₄-inhibitors;    -   (8) PAF-receptor antagonists;    -   (9) gold in the form of an aurothio group together with one or        more hydrophilic groups;    -   (10) immunosuppressive agents selected from the group consisting        of cyclosporine, azathioprine, and methotrexate;    -   (11) anti-inflammatory glucocorticoids;    -   (12) penicillamine;    -   (13) hydroxychloroquine;    -   (14) anti-gout agents including colchicine; xanthine oxidase        inhibitors including allopurinol; and uricosuric agents selected        from probenecid, sulfinpyrazone, and benzbromarone;-   C.) where older mammals are being treated for disease conditions,    syndromes and symptoms found in geriatric mammals, said inhibitory    compound is administered in combination with one or more members    independently selected from the group consisting essentially of:    -   (1) cognitive therapeutics to counteract memory loss and        impairment;    -   (2) anti-hypertensives and other cardiovascular drugs intended        to offset the consequences of atherosclerosis, hypertension,        myocardial ischemia, angina, congestive heart failure, and        myocardial infarction, selected from the group consisting of:        -   a. diuretics;        -   b. vasodilators;        -   c. β-adrenergic receptor antagonists;        -   d. angiotensin-II converting enzyme inhibitors            (ACE-inhibitors), alone or optionally together with neutral            endopeptidase inhibitors;        -   e. angiotensin II receptor antagonists;        -   f. renin inhibitors;        -   g. calcium channel blockers;        -   h. sympatholytic agents;        -   i. α₂-adrenergic agonists;        -   j. α-adrenergic receptor antagonists; and        -   k. HMG-CoA-reductase inhibitors            (anti-hypercholesterolemics);-   (3) antineoplastic agents selected from antimitotic drugs such as    the vinca alkaloids including but not limited to vinblastine and    vincristine;-   (4) growth hormone secretagogues;-   (5) strong analgesics;-   (6) local and systemic anesthetics; and-   (7) H₂-receptor antagonists, proton pump inhibitors, and other    gastroprotective agents.

The term “treating”, as used herein, refers to reversing, alleviating,inhibiting the progress of, or preventing the disorder or condition towhich such term applies, or one or more symptoms of such disorder orcondition. The term “treatment”, as used herein, refers to act oftreating, as “treating” is defined immediately above.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding equivalents of thefeatures shown and described, or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention claimed. Moreover, any one or more features of any embodimentof the invention may be combined with any one or more other features ofany other embodiment of the invention, without departing from the scopeof the invention. For example, the features of the anti-inflammatorycompounds of the present invention are equally applicable to the methodsof treating disease states and/or the pharmaceutical compositionsdescribed herein. All publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

EXAMPLES Example 1

Various pyrazolopyrimidine compounds were tested for anti-inflammatoryproperties using the methods detailed below.

Cytokines, Antibodies, and Reagents

Recombinant human interferon-γ (IFN-γ), TNF-α, interleukin (IL)-1β, andIL-4 were from R&D Systems (Minneapolis, Minn.). Histamine was fromSigma (St. Louis, Mo.). Mouse antibodies were obtained from commercialsources: murine IgG and anti-human vascular endothelial growth factorreceptor-2 (VEGFR2) (mIgG1; Sigma), anti-human tissue factor (mIgG1;Calbiochem, San Diego, Calif.), anti-human intercellular adhesionmolecule-1 (ICAM-1) (mIgG1; Beckman Coulter, Fullerton, Calif.), andanti-human E-selectin (mIgG1; HyCult Biotechnology, Uden, TheNetherlands). Mouse antibodies against human vascular cell adhesionmolecule-1 (VCAM-1) (mIgG1), HLA-DR (mIgG2a), CD3 (mIgG1), CD40 (mIgG1),CD69 (mIgG1), MIG (mIgG1), MCP-1 (mIgG1), CD14 (mIgG1), IL-1α (mIgG1),P-selectin (mIgG1), DAF (mIgG2a), urokinase-type plasminogen activatorreceptor (uPAR) (mIgG1), and CD38 (mIgG1) were obtained from BDBiosciences (San Jose, Calif.). Mouse antibodies against eotaxin-3(mIgG1), IL-8 (mIgG1), and M-CSF (mIgG1) were obtained from R&D Systems.Staphylococcal enterotoxin B, toxic shock syndrome toxin-1(staphylococcal enterotoxin F) from S. aureus (collectively calledsuperantigen; SAG), and lipopolysaccharide (LPS) from Salmonellaenteritidis were obtained from Sigma.

Human umbilical vein endothelial cells (HUVEC) were cultured asdescribed in Kunkel et al., 2004, FASEB J 18:1279-1281. Peripheral bloodmononuclear cells (PBMC) were prepared from buffy coats (Stanford BloodBank, Stanford, Calif.) by centrifugation over Hisopaque-1077 (Sigma).Four assay systems, 3C, 4H, SAG, and LPS, were used. For the 3C system,HUVEC were cultured for 24 h in microtiter plates (Falcon; BDBiosciences), in the presence of cytokines IL-1β (1 ng/ml), TNF-α (5ng/ml), and IFN-γ (20 ng/ml). For the 4H system, HUVEC were cultured inthe presence of IL-4 (5 ng/ml) and histamine (10 μM). For the SAGsystem, HUVEC were cultured with PBMC (7.5×10⁴) and SAG (20 ng/ml). Forthe LPS system, HUVEC were cultured with PBMC (7.5×10⁴) and LPS (2ng/ml). Compounds were added 1 h before stimulation and were presentduring the entire 24-h stimulation period. Cell-based enzyme-linkedimmunosorbent assay (ELISAs) were carried out as described. (See Kunkelet al., 2004, FASEB J18:1279-1281.)

Data Analysis

Mean optical density values for each parameter measured by ELISA werecalculated from triplicate samples per experiment. Well-to-wellcoefficients of variance range from 1 to 12%, depending on the parametermeasured, and average 5% across all controls. Day-to-day variability fora given readout, system, and treatment is the greatest contributor tothe overall variability (ranging from 10 to 60% of the totalvariability), but is controlled for by using a prediction envelope togive the error boundaries for all the measurements simultaneously,consistent with our multivariate analysis approach. The envelopeestimates the variability of the measurements around the mean (all dataare centered). By combining similar measurements from multipleexperiments, overall error measures are established while eliminatingthe specific bias of each experiment. Extensive studies have beenperformed concerning the number of repeats required for correctlyclassifying repeated profiles within given confidence limits leading tothe requirement for at least three replicate wells per treatment and atleast three independent repeats (unpublished observations).

Function Similarity Maps

Within each experiment, mean optical density values were used togenerate ratios between treated (e.g., compound or siRNA) and matchedcontrol (e.g., media or dimethyl sulfoxide) parameter values. Thesenormalized parameter ratios were then log 10 transformed. Log expressionratios were used in all Pearson correlation calculations. Correlationswere visualized in two dimensions by multidimensional scaling using AT&TGraphViz software. Distances between compounds are representative oftheir similarities, and lines are drawn between compounds whose profilesare similar at a level not due to chance. Significant correlations weredetermined by (a) identifying the number of correlations that exceed agiven threshold in the observed Pearson correlation distribution of theprofiles, (b) calculating the average number of Pearson correlationsthat exceed this threshold using correlations calculated from randomizeddata made by permuting the empirical profiles multiple times, (c)reselecting the Pearson correlation threshold to minimize the falsedetection rate (FDR) (the FDR provides the probability that asignificant correlation is a false positive), and (d) applying thiscutoff Pearson correlation value to the correlations betweenexperimental profiles. This ensures that for a 5% FDR, 95% of thecorrelations derived from the experimental profiles are not due tochance.

The results are presented in Table 1 below. Structure 13 in the Table isPP1 and not a compound of the invention. A “+” means that a compound wasnot active in the assay at a concentration of 5 micromolar. In oneembodiment, the compounds of the invention are compounds other thancompound nos. 1-57 in Table 1, below. In one embodiment, the methods ofthe invention are practiced with a compound shown in Table 1 other thanPP1. In one embodiment, the methods of the invention are practiced witha compound in Table 1 that is identified as “active” in the Activitycolumn. In one embodiment, the methods of the invention are practicedwith a compound other than compound nos. 1-57 in Table 1, below.

TABLE 1 No Compound Activity 1.

+ 2.

+ 3.

+ 4.

+ 5.

+ 6.

active 7.

active 8.

active 9.

active 10.

active 11.

active 12.

active 13.

active 14.

+ 15.

active 16.

+ 17.

active 18.

+ 19.

active 20.

+ 21.

+ 22.

active 23.

+ 24.

+ 25.

+ 26.

+ 27.

+ 28.

+ 29.

+ 30.

active 31.

active 32.

+ 33.

+ 34.

+ 35.

+ 36.

active 37.

+ 38.

+ 39.

active 40.

+ 41.

active 42.

+ 43.

+ 44.

+ 45.

+ 46.

+ 47.

active 48.

active 49.

+ 50.

active 51.

+ 52.

active 53.

active 54.

active 55.

+ 56.

+ 57.

+ 58.

+ 59.

active 60.

active 61.

+ 62.

active 63.

active 64.

active 65.

active 66.

active 67.

active 68.

+ 69.

+ 70.

+ 71.

+ 72.

active 73.

+ 74.

+ 75.

+ 76.

active 77.

active 78.

active 79.

active 80.

active 81.

active 82.

active 83.

active 84.

active 85.

active 86.

active 87.

active 88.

active 89.

active

In Table 1, the term “active” indicates that at least 3 parameters (inthe 4 systems tested (3C, 4H, SAG, and LPS)) simultaneously showedactivity falling outside a 99% prediction envelope, as described above.

Table 2, below, shows certain compounds that did not exhibit activity inthe systems tested at a concentration of 10 micromolar. In oneembodiment, the compounds of the invention are compounds other than thecompounds shown in Table 2. In one embodiment, the methods of theinvention are practiced with a compound other than a compound shown inTable 2, below.

TABLE 2 No Compound 1.

2.

3.

4.

5.

6.

Example 2

The following compounds provide illustrative characterization data forcertain pyrazolopyrimidine compounds. All starting materials andsynthetic reagents were purchased from commercial suppliers unlessotherwise noted. Acid chlorides that were not readily commerciallyavailable were synthesized by treating the corresponding carboxylicacids with excess oxalyl chloride and catalytic DMF in diethyl ether, asdescribed in Ward and Rhee, 1991, Tetrahedron Lett. 32:7165-7166. Usefulprotocols were adapted from Hanefeld et al. 1996, J. Chem. Soc., PerkinTrans. 1, 1996, 1545-1552. One skilled in the art will immediatelyrecognize that any differences in naming convention is not intended tolimit the illustrative value of the examples disclosed herein.

1-tert-Butyl-3-(2,5-dimethylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.79 (s, 9H), 2.20 (s, 3H), 2.25(s, 3H), 4.22 (s, 2H), 4.94 (s, 2H), 6.81 (s, 1H), 6.98 (d, 1H), 7.08(d, 1H), 8.23 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 19.3, 21.0, 29.2,33.1, 59.9, 100.9, 128.1, 129.1, 130.9, 133.6, 135.9, 136.3, 140.9,154.3, 154.6, 157.7; HRMS (EI) molecular ion calculated for C₁₈H₂₃N₅309.19535, found 309.19386.

1-tert-Butyl-3-(3,5-dimethylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.80 (s, 9H), 2.25 (s, 6H), 4.20(s, 2H), 5.00 (s, 2H), 6.80 (s, 2H), 6.88 (s, 1H), 8.22 (s, 1H); ¹³C NMR(CDCl₃, 100 MHz) δ 21.3, 29.2, 35.1, 59.9, 100.7, 126.2, 129.0, 138.0,138.9, 141.3, 154.4, 154.7, 157.7; HRMS (EI) molecular ion calculatedfor C₁₈H₂₃N₅ 309.19535, found 309.19439.

1-tert-Butyl-3-(3,4-dichlorobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.79 (s, 9H), 4.24 (s, 2H), 5.07(s, 2H), 7.02 (dd, 1H), 7.29 (d, 1H), 7.37 (d, 1H), 8.26 (s, 1H); ¹³CNMR (CDCl₃, 100 MHz) δ 29.1, 34.3, 60.2, 100.6, 127.6, 130.2, 131.0,131.4, 133.2, 138.4, 139.2, 154.6, 154.7, 157.5; HRMS (EI) molecular ioncalculated for C₁₆H₁₇N₅Cl₂ 349.08610, found 349.08621.

1-tert-Butyl-3-(4-chlorobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.77 (s, 9H), 4.25 (s, 2H), 5.16(br s, 2H), 7.11 (d, J=8 Hz, 2H), 7.27 (d, J=8 Hz, 2H), 8.22 (s, 1H);¹³C NMR (CDCl₃, 100 MHz) δ 29.1, 34.5, 60.0, 100.6, 129.3, 129.6, 133.1,136.6, 140.1, 154.5, 154.6, 157.6; HRMS (EI) molecular ion calculatedfor C₁₆H₁₈N₅Cl 315.12507, found 315.12545.

1-tert-Butyl-3-(4-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.78 (s, 9H), 3.76 (s, 3H), 4.22(s, 2H), 4.91 (br s, 2H), 6.84 (d, J=9 Hz, 2H), 7.11 (d, J=9 Hz, 2H),8.22 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 29.2, 34.3, 55.2, 59.9, 100.6,114.6, 129.4, 130.0, 141.4, 154.5, 154.7, 157.6, 158.7; HRMS (EI)molecular ion calculated for C₁₇H₂₁N₅O 311.17461, found 311.17454.

1-tert-Butyl-3-(2-methoxybenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.77 (s, 9H), 3.89 (s, 3H), 4.25(s, 2H), 5.77 (br s, 2H), 6.89 (m, 2H), 7.18 (m, 2H), 8.20 (s, 1H); ¹³CNMR (CDCl₃, 100 MHz) δ 27.4, 29.2, 55.5, 59.8, 100.4, 110.8, 121.5,126.5, 128.0, 130.6, 141.1, 154.3, 154.3, 155.7, 158.0; HRMS.

1-tert-Butyl-3-(2-chlorobenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.78 (s, 9H), 4.38 (s, 2H), 5.41(br s, 2H), 7.00 (dd, J₁=7 Hz, J₂=2 Hz, 1H), 7.15 (m, 2H), 7.39 (dd,J₁=8 Hz, J₂=2 Hz, 1H), 8.22 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 29.1,31.9, 60.0, 100.7, 127.4, 128.4, 129.6, 130.1, 133.3, 135.6, 139.4,154.4, 154.5, 157.7; HRMS (EI) molecular ion calculated for C₁₆H₁₈N₅Cl315.12507, found 315.12449.

1-tert-Butyl-3-(2-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.77 (s, 9H), 2.30 (s, 3H), 4.25(s, 2H), 5.09 (br s, 2H), 6.98 (d, J=8 Hz, 1H), 7.14 (m, 3H), 8.20 (s,1H); ¹³C NMR (CDCl₃, 100 MHz) δ 19.8, 29.1, 33.1, 59.9, 100.9, 126.7,127.3, 128.3, 130.8, 136.1, 136.8, 140.6, 154.3, 154.5, 157.8; HRMS (EI)molecular ion calculated for C₁₇H₂₁N₅ 295.17970, found 295.17922.

1-tert-Butyl-3-(4-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.79 (s, 9H), 2.30 (s, 3H), 4.24(s, 2H), 4.94 (br s, 2H), 7.08 (d, J=8 Hz, 2H), 7.11 (d, J=8 Hz, 2H),8.21 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 21.0, 29.2, 34.8, 59.9, 100.6,128.3, 130.0, 135.1, 136.9, 141.3, 154.5, 154.7, 157.7; HRMS (EI)molecular ion calculated for C₁₇H₂₁N₅ 295.17970, found 295.18068.

1-tert-Butyl-3-cyclopentylmethyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.White powder; ¹H NMR (CDCl₃, 400 MHz) δ 1.28 (m, 2H), 1.53 (m, 2H), 1.65(m, 2H), 1.71 (m, 2H), 1.73 (s, 9H), 2.28 (m, 1H), 2.86 (d, J=8 Hz, 2H),5.69 (br s, 2H), 8.25 (s, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 25.0, 29.2,32.4, 35.2, 39.7, 59.7, 100.5, 142.1, 153.0, 154.0, 158.0; HRMS (EI)molecular ion calculated for C₁₅H₂₃N₅ 273.19535, found 273.19565.

4-Amino-1-tert-butyl-3-(1′-naphthyl)pyrazolo[3,4-d]pyrimidine: whitepowder; ¹H NMR (270 MHz, CDCl₃) δ 1.92 (s, 9H), 5.04 (m, 2H), 7.43-7.73(m, 4H), 7.92-8.02 (m, 3H), 8.34 (s, 1H); HRMS (EI) molecular ioncalculated for C₁₉H₁₉N₅ 317.16427, found 317.16247.

4-Amino-1-tert-butyl-3-(2′-naphthyl)pyrazolo[3,4-d]pyrimidine: whitepowder; ¹H NMR (270 MHz, CDCl₃) δ 1.88 (s, 9H), 5.55 (m, 2H), 7.56-8.00(m, 6H), 8.16 (s, 1H), 8.39 (s, 1H); HRMS (EI) molecular ion calculatedfor C₁₉H₁₉N₅ 317.16427, found 317.16359.

4-Amino-1-tert-butyl-3-(m-phenoxyphenyl)pyrazolo[3,4-d]pyrimidine: whitepowder; ¹H NMR (270 MHz, CDCl₃) δ 1.83 (s, 9H), 5.61 (s, 2H), 7.08-7.49(m, 9H), 8.35 (s, 1H); HRMS (EI) molecular ion calculated for C₂₁H₂₁N₅O359.17483, found 359.17325.

4-Amino-1-tert-butyl-3-(m-benzyloxyphenyl)pyrazolo[3,4-d]pyrimidine:white powder; ¹H NMR (270 MHz, CDCl₃) δ 1.85 (s, 9H), 5.17 (s, 2H), 5.55(s, 2H), 7.10 (d, J) 8 Hz, 1H), 7.27-7.48 (m, 8H), 8.34 (s, 1H); HRMS(EI) molecular ion calculated for C₂₂H₂₃N₅O 373.19049, found 373.18833.

4-Amino-1-tert-butyl-3-(m-(2′,6′-dichloro)benzyloxyphenyl)pyrazolo[3,4-d]pyrimidine:white powder; ¹H NMR (270 MHz, CDCl₃) δ 1.85 (s, 9H), 5.36 (s, 2H), 5.74(s, 2H), 7.11-7.51 (m, 7H), 8.36 (s, 1H); HRMS (EI) molecular ioncalculated for C₂₂H₂₁Cl₂N₅O 441.11263, found 441.11050.

4-Amino-1-tert-butyl-3-piperonylpyrazolo[3,4-d]pyrimidine: white powder;¹H NMR (270 MHz, CDCl₃) δ 1.83 (s, 9H), 5.70 (s, 2H), 6.05 (s, 2H), 6.96(d, J) 8 Hz, 1H), 7.13-7.27 (m, 2H), 8.34 (s, 1H); HRMS (EI) molecularion calculated for C₁₆H₁₇N₅O₂ 311.13841, found 311.13777.

4-Amino-1-tert-butyl-3-(p-tert-butylphenyl)pyrazolo[3,4-d]pyrimidine:white powder; ¹H NMR (300 MHz, CDCl₃) δ 1.38 (s, 9H), 1.84 (s, 9H), 5.83(s, 2H), 7.58 (dd, J) 8 Hz, 12 Hz, 4H), 8.33 (s, 1H); HRMS (EI)molecular ion calculated for C₁₉H₂₅N₅ 323.21125, found 323.21024.

4-Amino-1-tert-butyl-3-(1′-naphthylmethyl)pyrazolo[3,4-d]pyrimidine:white powder; ¹H NMR (270 MHz, CDCl₃) δ 1.85 (s, 9H), 4.76 (s, 2H), 5.04(s, 2H), 7.19 (d, J) 6 Hz, 1H), 7.39 (t, J) 8 Hz, 1H), 7.55 (t, J) 4 Hz,2H), 7.79-7.92 (m, 2H), 8.20 (d, J) 8 Hz, 1H), 8.24 (s, 1H); HRMS (EI)molecular ion calculated for C₂₀H₂₁N₅ 331.17993, found 331.17951.

4-Amino-1-tert-butyl-3-(1′-naphthoxymethyl)pyrazolo[3,4-d]pyrimidine:beige powder; ¹H NMR (270 MHz, CDCl₃) δ 1.83 (s, 9H), 5.57 (s, 2H), 6.12(s, 2H), 7.07 (d, J) 7 Hz, 1H), 7.39-7.54 (m, 4H), 7.84 (d, J) 8 Hz,1H), 8.25 (d, J) 8 Hz, 1H), 8.35 (s, 1H); HRMS (EI) molecular ioncalculated for C₂₀H₂₁N₅O 347.17483, found 347.17408.

Example 3

The following example illustrates the ability of a compound useful inthe methods of the present invention to reduce neutrophil and monocyterecruitment, as well as to reduce the total number of leukocytes in amouse peritoneal inflammation model.

A single dose (10 mg/kg in 200 μl) of an anti-inflammatorypyrazolopyrimidine compound of the present invention (compound 30 inTable 1, above) was administered intraperionteally to a group of 6 micewhile another group of 6 mice received an intraperitoneal injection ofvehicle only. Fifteen minutes later, an inflammatory stimulus(thioglycollate, 200 μl) was administered intraperitoneally to eachmouse. Peritoneal lavage was collected at 4 and 24 hours subsequent tostimulus administration.

Inflammatory infiltrate was recovered by injection of 10 ml ofCa++/Mg++free PBS, massaging, and withdrawal. Total recovered cells werecounted on a hemacytometer. The results are presented in FIG. 2(average+/−SD of 6 mice).

Neutrophils, monocytes, lymophocytes and eosinophils were enumerated byanalyzing smears of the peritoneal wash stained with Wright-Geimsastain. The results are presented in FIG. 3 (average+/−SD of 6 mice).

1. A compound having the formula:

wherein R¹ is substituted or unsubstituted heteroaryl; R², R³, and R⁴are independently hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl; andL¹ is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted cycloalkylene or substituted or unsubstitutedheteroalkylene.
 2. A compound according to claim 1, wherein R² isR¹⁰-substituted C₁-C₂₀ alkyl, 2 to 20 membered substituted orunsubstituted heteroalkyl, R¹¹-substituted C₃-C₈ cycloalkyl,R¹¹-substituted C₃-C₈ heterocycloalkyl, R¹²-substituted heteroaryl, orR¹³-substituted aryl, wherein R¹⁰ is oxo, —OH, halogen, —CF₃, —NH₂, 2 to20 membered substituted or unsubstituted heteroalkyl, 3 to 7 memberedsubstituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heteroaryl, or R¹³-substituted aryl, R¹¹ is oxo, —OH,halogen, —CF₃, —NH₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, R¹² is—OH, halogen, —CF₃, —NH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl, andR¹³ is —OH; —NH₂, substituted or unsubstituted cycloalkyl, substitutedor unsubstituted heterocycloalkyl, substituted or unsubstitutedheteroaryl, or substituted or unsubstituted aryl.
 3. A compoundaccording to claim 1, wherein R¹ is R¹⁵-substituted heteroaryl, andfurther wherein R¹⁵ is —OH, halogen, —CF₃, —NH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.
 4. A compound according to claim 1, wherein R¹is substituted or unsubstituted fused-ring heteroaryl.
 5. A compoundaccording to claim 1, R³ and R⁴ are hydrogen
 6. A method for treating adisorder, said method comprising administering to a subject in needthereof a therapeutically effective amount of a compound according toclaim
 1. 7. The method of claim 6, wherein R¹ is R¹⁵-substitutedheteroaryl, and further wherein R¹⁵ is —OH, halogen, —CF₃, —NH₂,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.
 8. The method of claim 6,wherein R¹ is substituted or unsubstituted fused-ring heteroaryl.
 9. Themethod of claim 6, wherein R³ and R⁴ are hydrogen.
 10. The method ofclaim 6, wherein said disorder is an inflammatory process that occurs inresponse to infection, trauma, autoimmune disease, cardiovasculardisease, neoplasia, hyperplasia, addiction, infection, obesity, cellulardegeneration, apoptosis, or senescence, or differentiation.
 11. Themethod of claim 6, wherein said disorder is selected from the groupconsisting of vasculitis, multiple sclerosis, diabetes, inflammatorybowel disease, psoriasis, rheumatoid arthritis, Crohn's disease,ulcerative colitis, asthma, stroke, atherosclerosis, and lupus.
 12. Themethod of claim 6, wherein said disorder is selected from the groupconsisting of headaches, bronchitis, menstrual cramps, tendonitis,gastritis, vascular diseases, uveitis, Sjogren's disease, sclerodoma,nephrotic syndrome, swelling occurring after injury, myocardialischemia, fever, common cold, dysmenorrhea, emphysema, acute respiratorydistress syndrome, chronic obstructive pulmonary disease, Alzheimer'sdisease, organ transplant toxicity, cachexia, allergic reactions,allergic contact hypersensitivity, cancer, tissue ulceration, pepticulcers, regional enteritis, ulcerative colitis, diverticulitis,recurrent gastrointestinal lesion, gastrointestinal bleeding,coagulation, synovitis, gout, ankylosing spondylitis, restenosis,periodontal disease, epidermolysis bullosa, osteoporosis, loosening ofartificial joint implants, aortic aneurysm, periarteritis nodosa,congestive heart failure, myocardial infarction, cerebral ischemia, headtrauma, spinal cord injury, neuralgia, neuro-degenerative disorders,autoimmune disorders, Huntington's disease, Parkinson's disease,migraine, depression, peripheral neuropathy, pain, gingivitis, cerebralamyloid angiopathy, nootropic or cognition enhancement, amyotrophiclateral sclerosis, multiple sclerosis, ocular angiogenesis, cornealinjury, macular degeneration, conjunctivitis, abnormal wound healing,muscle or joint sprains or strains, tendonitis, skin disorders,myasthenia gravis, polymyositis, myositis, bursitis, burns, diabetes,tumor invasion, tumor growth, tumor metastasis, corneal scarring,scleritis, immunodeficiency diseases, sepsis, premature labor,hypoprothrombinemia, hemophilia, thyroiditis, sarcoidosis, Behcet'ssyndrome, hypersensitivity, kidney disease, Rickettsial infections,Protozoan diseases, reproductive disorders, and septic shock.